東 京 医 科 大 学 雑 誌
─278─ 第78巻 第3号
（ ）3
り小胞体ストレスの評価を、Annexin V/PI 染色細胞 のフローサイトメトリーおよび、ギムザ染色による 形態観察により細胞死の評価を行った。
＜結果・考察＞ A549 細胞において、FTY は
Lapおよび
Sorの殺細胞効果を顕著に増強したが、Dab および
Vemとの併用効果は小さかった。FTY と
Lapお よ び
Sorと の 併 用 効 果 は、 膵 癌 細 胞 株
BxPC-3および
PANC-1でも同様に観察され、特に
BxPC-3細胞における殺細胞効果の増強は、カスパー ゼ阻害剤
z-VAD-fmkを添加することで抑制された。
さらに
BxPC-3細胞では、
FTYと
Lapとの併用によっ
て、
Annexin V陽性細胞数の増加およびアポトーシ
ス特有の核の断片化、また、小胞体ストレス応答遺 伝子の発現増加および小胞体の膨化が観察された。
以上のことから、FTY と
Lapとの併用は、小胞体 ストレス負荷の増大を介して、BxPC
-3細胞のアポ トーシス様細胞死を誘導することが示唆された。
5.
Programmed cell death 2 forms coinhibitory mi- croclusters that directly attenuate T cell receptor signaling by recruiting phosphatase SHP2
（研究生
:免疫学、慶應義塾大学呼吸器内科）
○竹原 朋宏
（研究生
:免疫学）
若松 英、町山 裕亮、矢那瀬紀子 豊田 博子、古畑 昌枝、西 航 横須賀 忠
Background : Anti-PD-1 antibodies have made tremendous therapeutic effects on advanced or recurrent non-small cell lung cancer. However, the expression level of PD-L1 in tumor tissue which is the biomarker in the clinical setting is not necessarily correlated with their efficacy. In recent reports, some kinds of tumors express PD-L2 which is another ligand of PD-1. It is possible that the binding between PD-L2 and PD-1 is contributing to this mechanism. Because we intended to analyze the microstructural basis of immunological synapse, we used a system of a planar bilayer incorporated with glycophosphatidylinositol
（GPI ）
-anchored intercellular adhesion molecule 1（
ICAM-1）
and major histocompatibility complex（MHC）
class II（I
-Ek）loaded with a moth cytochrome c
（MCC）
peptide.Using our unique imaging analysis, we found the “TCR microcluster”, where TCR proximal signaling molecules are recruited. Here, we investigated the PD-1-PD-L2 pathway and PD-L1 vs PD-L2 competition toward PD-1 binding using this dynamic imaging technique.
Methods : We established tumor cell line
（
BHK）
highly expressing murine PD-L2（mPD
-L2）-GPI and purified mPD-L2-GPI by affinity column with anti-PD-L2.Primary CD4+ T cell isolated from AND-Tg mice in Rag2−/− Pdcd1−/− background and T cell hybridoma expressing AND-TCR
（
2D12）
were retrovirally transduced with PD-1-GFP or PD-1-HaloTag and GFP- SHP1/2. We used planar bilayers of mPD-L2-GPI, mICAM-1 and I-Ek loaded with MCC peptide and observed different kinds of T cell by confocal microscopy.Results : We showed PD-1 is translocated to TCR microclusters and then accumulates at the central region of the immunological synapse in the presence of PD-L2.
We also confirmed the rapid and transient recruitment of SHP2, not SHP1, to PD-1 microclusters by PD-1-PD-L2 binding. Biochemical assays demonstrated that PD-L2 dephosphorylates TCR downstream signaling molecules, resulting in the reduction of IL-2 production by forming PD-1-PD-L2 microclusters.
In the presence of both PD-L1 and PD-L2, PD-L2 accumulations toward PD-1 microclusters are stronger than those of PD-L1. We also confirmed the correlation between imaging results and IL-2 production.
Conclusions : Our results indicate that the function of PD-L1 and PD-L2 is the same in the point of suppressing TCR signaling by the recruitment of SHP2 for the initiation of T cell activation.
